Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility.
Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.
Pubmed ID: 11387207 RIS Download
Amino Acid Substitution | Antigens, CD29 | Breast Neoplasms | Cell Membrane | Cell Movement | Chromones | Cytoskeletal Proteins | Enzyme Inhibitors | Female | Green Fluorescent Proteins | Humans | Isoenzymes | Kinetics | Luminescent Proteins | Microscopy, Confocal | Morpholines | Mutagenesis, Site-Directed | Phorbol 12,13-Dibutyrate | Phosphatidylinositol 3-Kinases | Phosphoproteins | Phosphorylation | Phosphothreonine | Protein Kinase C | Protein Kinase C-alpha | Recombinant Fusion Proteins | Tumor Cells, Cultured | Wound Healing